Effective immunoprecipitation (IP) of cell cycle proteins Cyclin D, Cyclin E, Cyclin B and Cdk1

U2OS (human osteosarcoma) cells were synchronized at G0 followed by growth in 20% fetal bovine serum for 4, 6 and 18 hours prior to harvest. The cells were lysed in IP Lysis/Wash Buffer, and 0.75 mg of lysate (per sample) was incubated with anti-Cyclin D (rabbit polyclonal), anti-Cyclin E (mouse IgG1), anti-Cyclin B (mouse IgG1), or anti-Cdk1 (rabbit polyclonal) antibodies overnight at 4°C. The Pierce Protein A/G Magnetic Beads were added (50 µL each) to a 96 deep-well plate and immunoprecipitations were performed using the KingFisher Flex. Eluted sample volumes of 5 µL, 10 µL and 20 µL were resolved by SDS-PAGE and analyzed by western blot.

U2OS (human osteosarcoma) cells were synchronized at G0 followed by growth in 20% fetal bovine serum for 4, 6 and 18 hours prior to harvest. The cells were lysed in IP Lysis/Wash Buffer, and 0.75 mg of lysate (per sample) was incubated with anti-Cyclin D (rabbit polyclonal), anti-Cyclin E (mouse IgG1), anti-Cyclin B (mouse IgG1), or anti-Cdk1 (rabbit polyclonal) antibodies overnight at 4°C. The Pierce Protein A/G Magnetic Beads were added (50 µL each) to a 96 deep-well plate and immunoprecipitations were performed using the KingFisher Flex. Eluted sample volumes of 5 µL, 10 µL and 20 µL were resolved by SDS-PAGE and analyzed by western blot.

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